摘要： 电子克隆(In silicocloning)是随着基因组计划和EST计划实施而发展起来的利用生物信息学手段进行基因克隆的新方法。根据物种间同源基因相对保守的特点，以拟南芥(Arabidopsis thaliana)吡哆醇生物合成蛋白cDNA序列为信息探针，对大豆(Glycine max)EST数据库进行同源搜索和序列拼接，获得了1280 bp长的大豆吡哆醇生物合成蛋白的基因序列(GenBank登陆号为DQ139265)。经过RT-PCR扩增、基因组PCR扩增、分子克隆和序列分析验证，结果表明与电子克隆序列完全一致。该基因具有完整的开放阅读框架(ORF，20～955 bp)，编码311个氨基酸。通过与水稻、日本百脉根、烟草、截形苜蓿等物种的吡哆醇生物合成蛋白序列比对，发现该基因具有高度的保守性。表明根据物种间同源基因序列，对跨物种间EST数据库进行同源检索、筛选、拼接，是克隆基因的有效途径。

关键词: 电子克隆, EST数据库, RT-PCR, 基因组PCR, 大豆, 吡哆醇生物合成蛋白

Abstract: In silico cloning is developing with the project of genomic and EST. It is a new method of gene-cloning through bioinformatics. A query probe from the cDNA sequence of Arabidopsis thaliana pyridoxine biosynthesis protein was designed to blast Glycine max EST database according to the relative conservation of homologous genes and a soybean PDX gene cDNA of 1280 bp was obtained (GenBank Accession, DQ139265). This sequence was confirmed by RT-PCR, genomic PCR, molecular cloned and sequenced. It contained a complete ORF, from 20 to 955 bp, encoded 311 amino acids, and conserved with Arabidopsis thaliana, Oryza sativa, Lotus corniculatus var. japonicus, Nicotiana tabacum, Medicago truncatula and Triticum aestivum. The results showed that it was an efficient technique to clone new genes by searching EST database with homologous gene of model species.

Key words: In silico cloning, EST database, RT-PCR, Genomic PCR, Glycine max, Pyridoxine biosynthesis(protein)

中图分类号: 

• S565.1